DNA Replication and Repair Flashcards
What is the normal glutamine repeat allowance?
less than 28 CAG repeats
what is an insertion?
extra nucleotide is added, and can change the reading frame and cause a FRAMESHIFT mutation
what is heterochromatin?
condensed dna in the center or end of a chromosome
no expressed genes
what is primase?
RNA polymerase, it synthesizes a short RNA primer with a 3’ OH group
third step of base excision repair?
DNA polymerase fills in the gap
What is the reaction of the replication machinery to depurination?
the machinery can skip to the next complete nucleotide, producing a nucleotide deletion in the newly synthesized strand
what is FEN1?
flap endonuclease 1 -processes the 5’ end of Okazaki fragments to allow for ligation - removes 5’ “flaps” that hang off d/t unpaired bases
What sequences are repeated usually in telomeres?
TTAGGG
function of Ku70/80
acts as a scaffold on the ends of the broken DNA, guides DNA-PKcs to broken ends, protects ends from further degradation
what is the function of helicase?
sits at the front of the replication fork and uses energy of ATP hydrolysis to propel itself forward and pry open DNA double helix
what proteins are involved in nonhomologous end joining?
Ku70/80 (scaffold on the ends of the broken DNA, guides DNA-PKcs to broken ends, protects ends from further degradation)
DNA-PKcs (DNA dependent protein kinase catalytic subunit)
These two join together to make fully active DNA-PK
MRN, CtIP, artemis, ligase 4, XRCC4, XLF
what is RFC?
replication factor C - clamp loader protein - recognizes the single strand DNA binding protein (RPA) loads the clamp PCNA so that the front is oriented toward 3’ end (where dna pol delta will associate with PCNA)
large difference between NHEJ and homologous recombination?
NHEJ is prone to further mutation and error, homologous recombination is error free (it requires a homologous chromosome to be used as a template)
What is the function of DNA polymerase alpha?
it is complexed with primase to start the primer - initiates lagging strand synthesis
steps of the homologous recombination method
- 2 homologous chromosomes become aligned
- nuclease generates single stranded ends at the break (chews back one of the complementary strand)
- one of the single strands invades homologous DNA duplex by forming base pairs with complementary strand (makes a branch point where one strand from each duplex crosses) (makes a D loop comprised of overhanging 3’ invader and homologous chromosome)
- invading strand elongated by DNA polymerase, using the complementary strand as a template
- branch point migrates as base pairs holding together duplexes break and new ones form
- add’l dna synthesis and ligation complete repair
what does histone acetyltransferase do??
adds an acetyl group to lysine on the amino terminal tail of the histone
removes the positive charge and WEAKENS the bond between histone and DNA
therefore, opens region in dna for regulatory proteins to bind and increases gene expression
what does the thymidine analog of AZT do in DNA?
replaces thymidine during dna synthesis, therefore terminates the dna chain since there is no 3’ OH to add more nucleotides onto
what proteins are involved with mismatch repair?
proteins start with “MSH” or “MLH” the mismatch repair enzyme complex recognizes error, removes the segment with the mismatch, and DNA polymerase and ligase repair gap
ex. MSH6, MSH2, MLH1
which proteins are involved with excision repair?
these repair proteins start with XP - they recognize damage done to the DNA
ex. XPA, XPC, XPG, ETC
what end does DNA polymerase add nucleotides to?
3’ (3’ OH reacts with phosphate of incoming nucleotide)
2 general types of single nucleotide defect repair?
nucleotide excision repair and base excision repair, and mismatch repair
info about acyclovir?
what does this analog replace?
replaces guanosine triphosphate
third step of nucleotide excision repair?
newly sythesized segment is joined to 5’ end of remainder of original DNA strand by DNA ligase
what would occur if the mutation went unrepaired?
- substitution of base pair would be inserted (may be incorrect) 2. deletion of nucleotide pairs 3. stall of DNA replication (thymine dimer)
first step of nucleotide excision repair?
specific repair endonucleases cleave the abnormal chain and remove damaged region
pathology of Zellweger syndrome
mutations of peroxins (the proteins involved in formation of peroxisomes), leading to the accumulation of very long chain fatty acids
clinical characteristics: impaired brain development, liver and kidney lesions, and elevated very long chain fatty acids in the blood
when is mismatch repair performed?
REPLICATION ERROR: during DNA replication when an incorrect but normal base is incorporated into the chain (not normal watson-crick base pair) - there is a physical bulge that is recognized
what is depurination?
removing the purine base from a nucleotide (an OH is left in its place)
what is euchromatin?
contains expressed genes, is less condensed and more extended
what is the specific function of ATM?
is recruited by the MRN complex that recognizes the break, and activates P53.
Pathology of Tay-Sachs?
deficiency of hexosaminidase A in the lysosome, enzyme deficiency causes accumulation of gangliosides in the neurons, resulting in neuronal cell death
characterized by cherry red spot on retina
what is PCNA?
THE SLIDING CLAMP proliferating cell nuclear antigen, it is a DNA clamp that is a processivity factor for DNA pol delta, it encircles the dna and allows the polymerase to stay attached to DNA for longer amt of time
2 types of repair for double stranded breaks in DNA?
nonhomologous end joining and homologous recombination
What is a missense mutation?
results in codon that codes for a different amino acid
pathology of progeria
mutation in nuclear lamina gene Lamin A
causes cryptic splice site that mutates the final protein form into progerin.
progerin is not properly integrated into the lamina, causing a disfigured nucleus and altered genomic integrity
what is the cause of trinucleotide expansions?
slippage during DNA replication 1. newly synth strand dissociates from template strand 2. kink is formed 3. repeat sequences allow new strand to re-anneal in the wrong location, creating a duplication of that region
what is the function of P53
arrests the cell and gives it time to repair and recruit the proteins for repair, can also induce apoptosis
2 general causes of DNA mutations?
spontaneous, induced
what direction can DNA polymerase move in? ONLY
5-3
why can azidothymidine (AZT) treat retroviral infections?
the viral enzyme reverse transcriptase uses AZT triphosphate preferentially over cellular thymidine triphospate
what step occurs after the break recognition and resection in homologous recombination repair?
RPA coats the single strand DNA on the 3’ end, which summons Rad51 and Brca2. then Rad52 and Rad 54 load and these find identical DNA duplex and invade the sister chromatid
function of alpha-L-iduronidase
breaks down glycosaminoglycans
what is the function of Brca2?
binds the single strand DNA and directly interacts with the recombinase RAD51 to stimulate and maintain strand invasion
what proteins recognize double strand DNA breaks? and what do they do?
ATM, MRN complexes, nucleases
these recognize the break and begin to resect the 5’ end from the break
what is the most common mechanism for repairing double strand breaks in somatic cells?
non homologous end joining
characteristic of non homologous end joining vs homologous end joining?
NHEJ does not require a homologous chromosome as a template, so there may be mutations introduced
What is the cause of xeroderma pigmentosum?
the failure to repair thymine dimers after UV radiation exposure
what proteins bind to the exposed single DNA strands to keep the base pairs from coming back together? What is an example of one?
single strand binding proteins (SSB’s) RPA
pertinent information about acyclovir:
analog of what?
guanosine
which cells do not experience telomere shortening?
germ and stem cells
what is the function of dna polymerase epsilon?
leading strand synthesis
What binds to the ORC/MCM complex after they come together?
helicase - begins separating strands to form replication bubble
function of DNA-PKcs
finds Ku protein at the end of broken strands and forms DNA-PK active complex (kinase activity)
what are translocations?
a gross chromosomal rearrangement resulting from breaks in chromosomes, when the free ends of the DNA at the break points reseal with the free ends of a different broken chromosome
function of hexosaminidase A
breaks down ganglioside in the brain
what is the pathophysiology of xeroderma pigmentosum?
there is a defect in the nucleotide excision repair due to mutation in XP genes (XPA, XPB, XPC, XPD), so NER cannot remove UV damaged thymine dimers (or bases) and there is an accumulation of mutations
what are the 2 major types of DNA damage that absolutely need repair?
single nucleotide defects and double stranded breaks in DNA
when is base excisision repair specificlaly used?
it is used to correct spontaneous mutations introduced during replications due to deaminations and depurinations
pathology of Hurler syndrome
defect in alpha-L-iduronidase, resulting in accumulation of undigested glycosaminoglycans (GAGs) in the lysosome
clinically characterized by skeletal abnormalities, mental retardation, and nondegraded GAGs in urine
which cancer/syndrome is associated with mutations in mismatch repair proteins?
hereditary nonpolyposis colorectal cancer
Lynch Syndrome
what is a deletion?
loss of nucleotide, can change reading frame to result in frameshift mutation
why is acyclovir used to treat retroviral infections
retroviral thymidine kinase can convet the analog nucleoside into a nucleotide more efficiently
What is the function of the ORC?
origin recognition complex, binds to origin and recruits MCM
2 steps of nonhomologous end joining
- nucleases process the broken ends to form blunt ends (nucleotides are lost)
- the ends are brought together by enzymes and rejoined by DNA ligase
second step of base excision repair?
endonuclease cleaves the sugar phosphate strand at the site where the sugar phosphate backbone backs a base
when can homologous recombination occur in the cell cycle?
G2 and M phase, after replication
this is because the sister chromatids must be present (not prior to S phase)
second step of nucleotide excision repair?
gap made by first step is filled by DNA polymerase that adds deoxyribonucleotides one at a time to 3’, using intact complementary DNA strand as template
what pathway is defective in people with ataxia telangiectasia?
defective NHEJ and homologous recombination repair due to mutation in ATM
and are susceptible to agents that cause double stranded breaks
what is deamination?
spontaneous loss of an amino group from a cytosine, making the nitrogenous base a URACIL instead (O instead of NH2)
pathology of Lynch Syndrome?
mutations in MSH2 or MLH1
- the cells are unable to repair nucleotide mismatches
- afflicted person at increased risk for cancer
what is the reaction of the replication machinery to deamination?
machinery inserts an adenine when it encounters the uracil on the template strand
first step of base excision repair?
DNA glycosylase cleaves the N-glycosidic bond that joins that damaged base to the deoxyribose
what is AZT (azidothymidine) an analog of?
thymidine (has amino group instead of 3’ OH)
fourth step of base excision repair?
DNA ligase joins newly synthesized segment to the original DNA strand
what is a nonsense mutation?
results in a premature stop codon
when is nucleotide excision repair specifically used?
to removal thymine dimers and bulky adducts
what is the function of dna polymerase delta?
lagging strand synthesis
what is DNA ligase?
joins the 5’ phosphate end of one new DNA frag to the adjacent 3’ hydroxyl end of the next (fills in the gaps left by RNA primer removal) (makes a phosphodiester bond)
what does histone deacetylase do?
removes acetyl group from lysine tail, makes it more positive and increases the bonding of histones to dna, thus increasing condensation of dna and decreasing levels of gene expression
mechanism of benzo(a)pyrene induced mutation?
HIGHLY mutagenic, when oxidized by cytochrome P450, it forms bulky adducts with guanine residues in DNA
what does ultraviolet radiation do to DNA?
promotes covalent link between two pyrimidine bases forming pyrimidine (thymine) dimers
what stage does nonhomologous end joining occur in?
stage G1, when the cells are not in replication anymore
info about acyclovir:
what does this analog contain?
open chain structure instead of sugar molecule
types of point mutations?
- missense 2. nonsense 3. insertion/deletion
what is telomerase used for?
extends the DNA template to give space for RNA primer to be added for full replication - therefore, telomerase contains both proteins and RNA template (complimentary sequence to repeating sequence in telomere) IS A RNA-DEPENDENT DNA POLYMERASE
types of mutations?
- point mutations 2. trinucleotide expansions 3. chromosomal translocations
What is the function of topoisomerase?
make transient nicks in DNA backbone (phosphodiester bond of ONE strand) to relieve supercoiling, they reseal the DNA before falling off
What is Huntington’s Disease caused by, and what are the indicators?
trinucleotide expansion in the gene encoding protein huntingtin, when there are more than 40 CAG repeats (glutamine)
mechanism of xray induced mutation?
xray excites water in the cells, leading to hydroxyl radicals, which react with DNA, which alters the structure of the bases or cleaves the DNA strands (double strand break)
What is the function of the MCM?
binds to the ORC, unwinds double stranded DNA at these origins (helicase), recruits DNA polymerase, and initiates DNA asynthesis
what causes double stranded DNA breaks usually?
exposure to ionizing radiation (xrays and radioactive material)
what is RNAseH?
ribonuclease H, is an endoribonuclease that hydrolyzes the phosphodiester bonds of RNA when hybridized to DNA (aka cleaves RNA primer from the 5’ ends of okazaki fragments in a DNA/RNA hybrid) - does not digest single or double stranded DNA
correlation between cancer cells and telomeres?
cancer cells upregulate telomere activity and INCREASE the length of the telomere, adding to the immortality of cancer cells
what mutation do individuals with ataxia telangiestasia have?
mutation in ATM, so they have an impaired DNA damage response
which mutations can cause frameshift mutations?
insertions and deletions
what cancers are people with lynch syndrome more likely to develop? (8)
colorectal, endometrial, stomach, ovarian, ureter, brain, hepatobiliary tract, skin
what proteins are involved in NHEJ?
Ku70/80, DNA-PKcs, DNA-PK, MRN
proteins involved in homologous recombination?
ATM, MRN, nuclease, RPA, BRCA2, RAD51/52, p53
